Over the years investigators have attempted to harness the immune system for therapeutic use. Immunoglobulin (Ig) molecules which constitute an important part of the immune system are of great interest because they (1) react with a diverse family of ligands, (2) possess different effector functions and (3) are of great biological importance. Despite its potential, a persistent problem with immunoglobulin immunotherapy has been, among other problems, the toxic effect to normal cells of using antibodies which recognize both normal and diseased cells. This problem is far-reaching because the majority of antibodies presently available recognize a target located on both normal and diseased cells (Slavin-Chiorini, et al., Int. J. Cancer 53: 97–103 (1993)).
The constant region can promote cell death through antibody dependent cell medicated cytotoxicity (ADCC) or by complement dependent cytotoxicity (CDC). Despite the deletion of portions of the constant region, particularly the CH2 domain, the antigen binding function can be retained (D. Yelton, M. Scharf, Mutant monoclonal antibody with alterations in biological functions, J. Exp. Methods 156:1131–1148 (1982)).
Others have generated a CH2-deleted antibody (Mueller et al., Proc. Natl. Acad. Sci. USA 87: 5702–5705 (1990)). Their findings provide that the CH2-deleted antibody was cleared from the blood of tumor-bearing mice much faster than the corresponding intact antibody. Other in vivo findings also confirmed that a CH2-deleted antibody, designated ch14.18DCH2, is a potentially useful reagent for radioimmunodetection of human tumors because of its reduced immunogenicity, increased target specificity, and rapid clearance from circulation (Mueller et al., Proc. Natl. Acad. Sci. USA 87: 5702–5705 (1990).
Generally, whole antibody molecules are composed of two heavy (H) and two light (L) chains which are held together by covalent bonds (disulfide) and non-covalent interactions. Each chain contains a variable region (V) and a constant region (C). The variable regions at the amino termini of the two chains form the antigen binding region. The constant region of the H chain has three components or domains. The first constant region domain (CH1) interacts with the C region of the L chain through hydrophobic interactions and generally a disulfide bond, depending on isotype. The next C region stretch is the hinge-acting disulfide bonds stably introduced between two H chains. The second constant region domain (CH2) is adjacent to the hinge region. CH2 contains sequences important for effector functions of the antibody, such as the sequences responsible for complement fixation and Fc receptor binding. The third constant region domain (CH3) is located at the carboxyl terminus of the H chain, and is considered to play an important role in H chain assembly as well as some C region functions.
Today many antibodies in clinical trials are directed against tumor associated antigens. Most tumor associated antigens are not tumor specific but are also generally found on the cell surface of some normal, non-tumorigenic cells. The clinical use of some antibodies directed against tumor associated antigens are limited because of the toxicity associated with their use. Therefore, there is a need for methods of inhibiting toxicity associated with immunoglobulin use in the field of disease therapy (e.g., therapy for tumors, kidney disease, and the like) and in vivo diagnosis.
We addressed this need by discovering methods for inhibiting or reducing toxicity to normal cells generally associated with immunoglobulin immunotherapy or in vivo diagnosis, wherein the immunoglobulin recognizes both diseased and normal cells. Our discovery involves generating immunoglobulin molecules or Ig fusion proteins having structurally altered constant regions which inhibit or reduce immunoglobulin-induced toxicity.